As indicated above, the MN gene and protein are known by a number of alternative names, which names are used herein interchangeably. The MN protein was found to bind zinc and have carbonic anhydrase (CA) activity and is now considered to be the ninth carbonic anhydrase isoenzyme—MN/CA IX or CA IX [Opavsky et al., “Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships,” Genomics. 33: 480-487 (1996)]. According to the carbonic anhydrase nomenclature, human CA isoenzymes are written in capital Roman letters and numbers, whereas their genes are written in italic letters and Arabic numbers. Alternatively, “MN” is used herein to refer either to carbonic anhydrase isoenzyme IX (CA IX) proteins/polypeptides, or carbonic anhydrase isoenzyme 9 (CA9) gene, nucleic acids, cDNA, mRNA etc. as indicated by the context.
The MN protein has also been identified with the G250 antigen. Uemura et al. [“Expression of Tumor-Associated Antigen MN/G250 in Urologic Carcinoma: Potential Therapeutic Target,” J. Urol. 157 (4 Suppl.): 377 (Abstract 1475; 1997)] states: “Sequence analysis and database searching revealed that G250 antigen is identical to MN, a human tumor-associated antigen identified in cervical carcinoma (Pastorek et al., 1994).”
Zavada et al., International Publication No. WO 93/18152 (published Sep. 16, 1993) and U.S. Pat. No. 5,387,676 (issued Feb. 7, 1995) describe the discovery of the MN gene and protein. The MN gene was found to be present in the chromosomal DNA of all vertebrates tested, and its expression to be strongly correlated with tumorigenicity. In general, oncogenesis may be signified by the abnormal expression of CA IX protein. For example, oncogenesis may be signified: (1) when CA IX protein is present in a tissue which normally does not express CA IX protein to any significant degree; (2) when CA IX protein is absent from a tissue that normally expresses it; (3) when CA9 gene expression is at a significantly increased level, or at a significantly reduced level from that normally expressed in a tissue; or (4) when CA IX protein is expressed in an abnormal location. WO 93/18152 further discloses, among other MN-related inventions, MN/CA IX-specific monoclonal antibodies (MAbs), including the M75 MAb and the VU-M75 hybridoma that secretes the M75 MAb. The M75 MAb specifically binds to immunodominant epitopes on the proteoglycan (PG) domain of MN/CA IX.
Zavada et al., International Publication No. WO 95/34650 (published Dec. 21, 1995) provides in FIG. 1 the nucleotide sequence for a full-length MN cDNA [SEQ ID NO: 1] clone isolated as described therein, and the amino acid sequence [SEQ ID NO: 2] encoded by that MN cDNA. WO 95/34650 also provides in FIG. 3A-F a 10,898 base pair (bp) complete genomic sequence of MN [SEQ ID NO: 3], and in FIG. 6 the nucleotide sequence for the MN promoter [SEQ ID NO: 4]. Those MN cDNA, amino acid, genomic, and promoter sequences are incorporated by reference herein.
Zavada et al., International Publication No. WO 03/100029 (published Dec. 4, 2003) discloses among other MN-related inventions, MN/CA IX-specific MAbs that are directed to non-immunodominant epitopes, including those on the carbonic anhydrase (CA) domain of MN/CA IX. An example of such a MN/CA IX-specific MAb is the V/10 MAb, secreted from the V/10-VU hybridoma.
The MN protein is now considered to be the first tumor-associated carbonic anhydrase isoenzyme that has been described. The carbonic anhydrase family (CA) includes twelve catalytically active zinc metalloenzymes involved in the reversible hydration-dehydration of carbon dioxide: CO2+H20HCO3−+H+. CAs are widely distributed in different living organisms. The CAs participate in a variety of physiological and biological processes and show remarkable diversity in tissue distribution, subcellular localization, and biological functions, including pH regulation, CO2 and HCO3 transport, and water and electrolyte balance. [Parkkila and Parkkila, Scand J Gastroenterol., 31: 305-317 (1996); Potter and Harris, Br J Cancer, 89: 2-7 (2003); Wingo et al., Biochem Biophys Res Commun, 288: 666-669 (2001); Christianson and Cox, Ann Rev Biochem, 68: 33-57 (1999); Supuran et al., Curr Med Chem Cardiov Hemat Agents, 2: 51-70 (2004).] CA IX is a glycosylated transmembrane CA isoform with a unique N-terminal proteoglycan-like extension [Opavsky et al. (1996)]. Through transfection studies it has been demonstrated that CA IX can induce the transformation of 3T3 cells [Opavsky et al. (1996)].
Many studies, using the MN-specific monoclonal antibody (MAb) M75, have confirmed the diagnostic/prognostic utility of MN in diagnosing/prognosing precancerous and cancerous cervical lesions [Liao et al., Am. J. Pathol., 145: 598-609 (1994); Liao and Stanbridge, Cancer Epidemiology, Biomarkers & Prevention, 5: 549-557 (1996); Brewer et al., Gynecologic Oncology 63: 337-344 (1996)]. Immunohistochemical studies with the M75 MAb of cervical carcinomas and a PCR-based (RT-PCR) survey of renal cell carcinomas have identified MN expression as closely associated with those cancers and confirm MN's utility as a tumor biomarker [Liao et al. (1994); Liao and Stanbridge (1996); McKiernan et al., Cancer Res. 57: 2362-2365 (1997)]. In various cancers (notably uterine cervical, ovarian, endometrial, renal, bladder, breast, colorectal, lung, esophageal, head and neck and prostate cancers, among others), CA IX expression is increased and has been correlated with the microvessel density and the levels of hypoxia in some tumors [Koukourakis et al. (2001); Giatromanolaki et al. (2001)].
In tissues that normally do not express MN protein, CA IX positivity is considered to be diagnostic for preneoplastic/neoplastic diseases, such as, lung, breast and cervical precancers/cancers [Swinson et al. (2003); Chia et al. (2001); Loncaster et al. (2001)], among other precancers/cancers. Very few normal tissues have been found to express MN protein to any significant degree; those MN-expressing normal tissues include the human gastric mucosa and gallbladder epithelium, and some other normal tissues of the alimentary tract [Pastorekova and Zavada, “Carbonic anhydrase IX (CA IX) as a potential target for cancer therapy,” Cancer Therapy, 2: 245-262 (2004); Pastorekova et al., “Carbonic Anhydrase IX: Analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts,” Gastroenterology, 112: 398-408 (1997); Leppilampi et al., “Carbonic anhydrase isozymes IX and XII in gastric tumors,” World J Gastroenterol, 9: 1398-1403 (2003)].
MN/CA IX and Hypoxia
Recent studies have revealed that CA IX not only participates in cell adhesion and pH regulation, but also can be induced in hypoxia via the HIF-1 protein binding to the hypoxia-responsive element of the MN promoter [Svastova et al., Exp Cell Res, 290: 332-345 (2003); Wykoff et al., Cancer Res, 60: 7075-7083 (2000)]. Hypoxia is a common feature in solid tumors. It is a pathophysiologic consequence of a structurally and functionally disturbed microcirculation and the deterioration of oxygen diffusion conditions [Höckel and Vaupel (2001)]. Tumor hypoxia has long been known to be associated with poor survival in cancer patients, since it may contribute to the development of more malignant tumor phenotypes and increase tumor invasiveness and metastatic potential [Harris (2002)]. Hypoxia also has an important role in the development of resistance to chemotherapy and radiotherapy [Höckel et al. (1996)].
It is recognized that tumor cells under hypoxic conditions maintain a low extracellular pH (pHe) and a high intracellular pH [Svastova et al. (2004)]. This confers a survival advantage by possible prevention of tumor cell apoptosis and facilitates the local invasiveness of the tumor by breakdown of the extracellular matrix [Svastova et al. (2004)]. In addition, the acidic tumor microenvironment may reduce the uptake of drugs which are weak bases and hence ionized at acid pH [Gerweck and Seetharaman (1996); Raghunand et al., (1999); Raghunand and Gillies (2001)]. For example, under some circumstances, an acidic tumor environment may indicate against the use of anthracyclines.
MN/CA IX Hypoxic Regulation
The transcription of the MN gene is negatively regulated by wild-type von Hippel-Lindau tumor suppressor gene in transfected renal cell carcinoma cells [Ivanov et al., Proc Natl Acad Sci (USA), 95: 12596-12601 (1998)]. The protein product of the von Hippel-Lindau tumor suppressor gene interacts with the ubiquitin ligase complex that is responsible for targeting HIF-1α for oxygen-dependent proteolysis [Maxwell et al., Nature, 399: 271-275 (1999); Jaakkola et al., Science, 292: 468-472 (2001)]. Thus, low levels of oxygen lead to stabilization of HIF-1α, which in turn leads to the increased expression of MN [Wykoff et al. (2000)]. Areas of high expression of MN in cancers are linked to tumor hypoxia as reported in many cancers, and incubation of tumor cells under hypoxic conditions leads to the induction of MN expression [Wykoff et al. (2000); Koukourakis et al., Clin Cancer Res, 7: 3399-3403 (2001); Giatromanolaki et al., Cancer Res, 61: 7992-7998 (2001); Swinson et al., J Clin Oncoli, 21: 473-482 (2003); Chia et al., J Clin Oncol, 19: 3660-3668 (2001); Loncaster et al., Cancer Res, 61: 6394-6399 (2001)]. Expression of MN/CA IX is localized to the perinecrotic area of tumors and has been observed to start at a median distance of 80 μm from a blood vessel, where the oxygen tension drops to 1%, in head and neck squamous cell carcinoma [Beasley et al. (2001)].
MN/CA IX and Breast Cancer Therapy
It has been shown that MN/CA IX can acidify the pHe of tumor cells in a culture medium and downregulation reduces the survival of breast tumor cells under hypoxic conditions [Potter and Harris (2003)]. In three studies, the expression of MN/CA IX was associated with poor prognosis independent of the other commonly recognized prognostic parameters such as tumor (T) status, node (N) status, tumor grade, estrogen receptor (ER), and c-erbB2 expression in breast cancer patients [Chia et al. (2001); Bartosova et al. (2002); Span et al. (2003)]. All those studies involved heterogeneous patient populations submitted postoperatively to different treatment strategies (radiotherapy, chemotherapy, and endocrine therapy) or no therapy.
Primary chemotherapy administered to the breast cancer patients is a useful model to identify baseline features able to predict which patients are most likely to benefit from the cytotoxic treatment and is a way to study new biological markers in relation to the predictive information they provide. In addition, tumor biopsy specimens obtained in matched pair cases at diagnosis and definitive surgery provide valuable information on the interaction between biological markers and treatment.
For example, breast cancer patients are routinely tested for the presence or absence of the estrogen receptor in an attempt to predict whether the patients will be resistant or responsive to tamoxifen [Nolvadex®; AstraZeneca], a nonsteroidal antiestrogen that is currently the most widely used breast cancer treatment. Based on the current test, cancer patients who test positive for the presence of estrogen receptors (“ER positive”) are typically prescribed tamoxifen. However, a significant number of ER positive patients are in fact resistant to tamoxifen. Therefore, administration of tamoxifen to a patient who is resistant to its benefits may cause delay by preventing the patient from undergoing more effective treatments. Further, tamoxifen administration has been associated with an increased risk of endometrial cancer. An accurate determination of whether a patient will be susceptible or resistant to the antineoplastic effects of tamoxifen administration, before embarking on such a treatment course, would be a valuable diagnostic tool. Due in large part to the limited ability of clinical criteria to assess accurately an individual's risk, many patients continue to be overtreated or undertreated [Lyman et al. (2007)].
Commercialized multigene assays have been developed to predict clinical outcome for breast cancer. For example, the Oncotype DX™ diagnostic assay was recently developed by Genomic Health, and tests for 21 genes, including genes associated with proliferation, estrogen and HER-2 activity, invasion, as well as five control genes. This assay provides a recurrence score for lymph node negative breast cancer patients with estrogen receptor positive tumors that have received adjuvant tamoxifen [Paik et al., Breast Cancer Res. Treat., 82: S10 (2003)]. However, such a multigene test is expensive: the cost of Oncotype DX™ patient testing was estimated in 2005 as $3,450 per patient [Hornberger et al., Am J Manag Care, 11(5): 317 (2005)]. It would be useful to have an assay to predict clinical outcome for tamoxifen treatment of patients with breast cancer, that was relatively inexpensive and could be performed routinely, based on the detection of expression of only one gene, and which could be also performed by immunohistochemistry.
Disclosed herein are methods wherein MN overexpression is shown to be useful as a prognostic marker for estrogen receptor (ER) positive breast cancer, particularly for those patients who are treated with, or under consideration for treatment with, endocrine therapy, particularly tamoxifen therapy. MN positive expression in ER-positive breast cancer patients treated with tamoxifen was found to be a poor prognostic factor, and conversely MN negative expression was found to be a good prognostic factor. The prognostic methods disclosed herein detect MN overexpression, and can identify high-risk estrogen receptor positive breast cancer patients who could benefit from additional and/or alternative therapies, such as adjuvant chemotherapy or immunotherapy and MN-targeted therapies, among other appropriate therapies.